The present invention relates generally to methods and systems for hybridizing and/or incubating microarrays.
Microarrays are arrays of very small samples of purified DNA, protein, antibody, or small molecule target material arranged as a grid of up to hundreds or thousands of small spots immobilized onto a solid substrate. FIG. 1A is a top view of a typical microarray. The microarray substrate 10 is typically coated or derivatized uniformly over its top surface 12 to afford chemical or electrostatic binding of small droplets of the target material in solution. The droplets of target solution dry and bind to the top surface 12 of the substrate 10, forming target spots 5 that are is generally from tens to hundreds of microns in diameter. The target spots 5 form a spotted area 7 on the top surface 12 of the substrate 10. The spotted area 7 in the substrate 10 of FIG. 1A is rectangular and has a broken line drawn around it.
A microarray can be used to detect complementary probes. The immobilized target spots on the microarray substrate are exposed to complementary DNA, protein, antigen, or chemical probe samples in liquid solution. The probe materials in solution, which are generally derived from cells, bodily fluids, or combinatorial chemistry libraries, are labeled with fluorescent dyes. The probe materials bind at complementary target spots on the microarray, and the dyes allow for subsequent detection and measurement of the relative concentration of each species of complementary probe material at each target spot. Other detection schemes may be used aside from fluorescence, such as the use of radioactive markers, chemiluminescence, and surface plasmon resonance (SPR).
In some references relating to microarrays, the nomenclature for the immobilized spot material on the microarray substrate (called xe2x80x9ctargetxe2x80x9d material here) and the solution applied to the spots for selective binding assays (called xe2x80x9cprobexe2x80x9d material here) is reversed.
Through a process called hybridization, DNA probe material in solution selectively binds to target spots on the microarray substrate only where complementary bonding sites occur. Similarly, labeled protein probe material only binds selectively to target spots with specific complementary bonding sites; this process is called affinity binding and incubation in protein and antibody assays. Selective reactions of smaller organic or inorganic chemicals (small molecules) to one another or to proteins or DNA can occur in the same way. DNA hybridization and the terminology associated with DNA microarrays will be used throughout this specification, but it is to be understood that the same processes and effects apply to these other types of microarrays.
After the reaction between the probe material and the target material is allowed to occur, quantitative scanning in a fluorescent microarray scanner produces a pixel map of fluorescent intensities. This fluorescent pixel map can be analyzed by special purpose quantitation algorithms to reveal the relative concentrations of the fluorescent probe materials at each target spot on the microarray, thus indicating the level of gene expression, protein concentration, or the like present in the cells from which the probe materials were extracted.
The microarray substrate is generally made of glass that has been treated chemically to provide for molecular attachment of the target spot samples of microarray target material. The substrate 10 can also be made of plastic, silicon, ceramic, metal, or other rigid material. The microarray substrate 10 is also generally of the same size and shape as a standard microscope slide, about 25 mmxc3x9775 mmxc3x971 mm thick. The array area of target spots can extend to within about 1.5 mm of the edges of the substrate, although this array area can also be smaller. Typically, the target spots are approximately round. The target spot diameter can vary from about 50 microns to about 500 microns, depending on the dispensing or spotting technique used to apply the target spots to the microarray substrate. The center-to-center spacing between the target spots on the microarray substrate usually falls into the range of about 1.5 to 2.5 target spot diameters. The target spots are typically printed or xe2x80x9cspottedxe2x80x9d on the top surface of the substrate by pin-type spotting instruments which deposit droplets by a stamping process, where a small ( less than 1 nanoliter) amount of liquid from the wetted end of the pin is transferred to the top surface of the substrate. Alternately, piezo-electric dispensers can dispense drops onto a substrate""s activated surface (called the spotted area of the top surface here) in a manner similar to an ink-jet printer.
The protocols for producing the fluorescently labeled probe solutions can be fairly complex. For differential gene-expression DNA microarrays, exemplary probe preparation steps are:
Tissue or cell isolation
RNA extraction
RNA purification
Reverse transcription of RNA to cDNA
Attachment of the fluorescent label to all species of DNA the solution
Dye teminator cleanup
Addition of buffer to attain desired volume, concentration, pH, etc.
A common type of microarray is used for analyzing differential gene expression. Labeled probe material is prepared from each of two or more tissues or cell types; the RNA/cDNA extracted from each tissue type is labeled with a different dye. Then, the two or more labeled probes are mixed together and applied in solution form to the microarray. The probe mixture is kept in intimate contact with the immobilized target spots on the microarray for some number of hours, typically at a temperature above ambient temperature, to allow the complementary strands of DNA to come into contact with one another and to bind. This process is generally called xe2x80x9cincubation,xe2x80x9d and xe2x80x9chybridizationxe2x80x9d is used to refer to single-stranded DNA segments binding into a double-helix. In contrast, antibody-antigen assay incubation is often carried out at room temperature for times on the order of 5-60 minutes.
FIG. 1B shows a side view of a typical cover glass arrangement that has been used for reacting the probe material with target spots. In the arrangement of FIG. 1B, the microarray substrate 10 is placed on a work surface 14 with the top surface 12 of the microarray substrate 10 having the spotted area 7 facing up. A selected volume of liquid probe solution 16 is then placed as a thin layer on the top surface 12 where the spotted area 7 (not shown) is located, and a cover glass 18 is placed over the liquid probe solution 16. A typical volume of the liquid probe solution 16 is about 15-25 microliters. This small volume of liquid probe solution 16 is deposited on the spotted area 7 of the top surface 12 as a drop. The cover glass 18 placed on top of the drop of liquid probe solution 16 spreads the drop into a thin layer over the spotted area 7 of the top surface 12 with about the same dimensions as the cover glass 18. The layer of liquid probe solution 16 can be about 10-60 microns thick and is kept in place by the capillary effect of being sandwiched between two planar pieces of glass. The dimensions of the spotted area 7 on the top surface 12 and the cover glass 18 are usually smaller than the dimensions of the microarray substrate 10, but in some cases the spots, the cover glass 18 and the liquid probe solution 16 can cover the entire top surface 12 of the microarray substrate 10.
The microarray substrate 10 with liquid probe solution 16 and the cover glass 18 is then placed in a sealed chamber of some sort to prevent the probe from evaporating or drying during incubation. Specially designed hybridization chambers are available for this (a Telechem Hybridization Cassette, for example), but many researchers use common labware such as 50 ml centrifuge tubes or Copeland jars. Often, a laboratory wipe or other absorbent object soaked with water is placed into the hybridization chamber with the microarray and probe liquid to keep the humidity in the chamber near 100% to minimize drying of the probe liquid. Drying of the probe mixture leads to very high non-specific attachment of the fluorescent dye to the microarray, which in turn causes very high background fluorescent signals that may drown out the hybridization signals where drying has occurred.
The molecular event that causes a labeled molecule in the liquid probe solution 16 to bind to one of its immobilized complements on the top surface 12 requires that the two molecules be in intimate contact. With the cover glass method described in connection with FIG. 1B, diffusion is the only vehicle for molecular movement. In other methods, a stick-on cap can be affixed over a substrate with liquid probe solution, and then the liquid probe solution can be agitated during incubation by shaking the combination of the substrate and stick-on cap, or by pumping liquid to and from under the stick-on cap.
One embodiment of the invention is a method for incubating a liquid reagent with target spots on a first surface of a microarray substrate. In this embodiment, the liquid reagent is confined between a deformable cover and the surface of the substrate having the target spots. The deformable cover is then deformed by applying a force to the cover with a deflector. The force can vary in location of application, magnitude, or in a combination of magnitude and location of application. The deflector, which can be a roller, can apply a force to the deformable cover in different locations along the deformable cover, thus agitating the liquid reagent and aiding in incubation. Deformation of the deformable cover can be in either a top region or in a gasket of the cover.
In an alternative method for incubating reagents in accordance with the invention, a deformable cover is placed upon a mechanical support or a work surface. In one embodiment, the deformable cover is placed upside-down on the work surface. Liquid reagent is then placed on the deformable cover, either manually or automatically. The microarray substrate is then placed over the cover with the liquid reagent, thus forming a reaction chamber between the liquid reagent and the substrate. The microarray substrate and the deformable cover are then moved to agitate the liquid reagent. A force can be applied to the deformable cover with a deflector to agitate the liquid reagent in the reaction chamber. Upon application of the force, the deformable cover can deform to move the liquid reagent in the reaction chamber. Additionally, when the liquid reagent is placed in the cover, the amount of liquid reagent can be applied so that an air bubble remains within the reaction chamber upon application of the substrate over the deformable cover. Upon application of the force to the deformable cover, the air bubble can aid in the agitation of the liquid reagent.
Another embodiment of the invention is an apparatus for incubating a liquid reagent with target spots on a first surface of a microarray substrate. In this embodiment, the apparatus can include a deformable cover and a deflector. The deformable cover is adapted to seal the liquid reagent between the deformable cover and the first surface of the microarray substrate, thus forming a reaction chamber. The deflector is designed to apply a force to the deformable cover to agitate the liquid reagent within the reaction chamber.
Yet another embodiment of the invention is also an apparatus for incubating a liquid reagent with target spots on a first surface of a microarray substrate. In this embodiment, the cover includes a substantially rigid lid and a gasket that deforms more easily than the lid. A first actuator and a second actuator are used to apply forces to the cover, thus deforming the gasket of the cover. Upon alteration of the force produced by the first actuator and the force produced by the second actuator, the lid of the cover tilts, thus producing a flow of liquid reagent over the microarray substrate. This flow of liquid reagent can aid in the reaction during incubation. In one embodiment, the sum of the force produced by the first actuator and the force produced by the second actuator remains substantially constant as the two forces vary. In this manner, when one of the forces increases, the other force decreases by approximately the same magnitude. This substantially constant sum of the forces can ensure that a sufficient force remains on the cover to keep the seal formed between the gasket and the substrate so that liquid reagent does not escape during the incubation process.